Targeted CRISPR‐Cas9‐based gene knockouts in the model brown alga Ectocarpus
- 2 June 2021
- journal article
- research article
- Published by Wiley in New Phytologist
- Vol. 231 (5), 2077-2091
- https://doi.org/10.1111/nph.17525
Abstract
Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.Keywords
Funding Information
- Marine Alliance for Science and Technology for Scotland
- European Cooperation in Science and Technology (Phycomorph)
- Gordon and Betty Moore Foundation (Symbiosis in Aquatic Systems Initiative 4546891618)
- H2020 European Research Council (SexSea 638240)
- Natural Environment Research Council (NE/L013223/1)
- Horizon 2020 Framework Programme (ASSEMBLE PLUS 730984)
- Agence Nationale de la Recherche (ANR‐10‐BTBR‐04‐01, ANR‐19‐CE20‐0028‐01)
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